Universally pseudotyped retroviral compositions and methods

Abstract

Provided herein is a retroviral vector system that is useful for producing universal pseudotyped retroviruses for cell therapy and gene therapy.This vector system comprises an envelope plasmid and a packaging plasmid, and at least one of these plasmids encodes a binding moiety that directly binds to target cells via cell binding domain or indirectly binds to target cells via antibody binding domain.Also provided are related retroviral packaging cells, retroviruses, virus-like particles, and the methods for their production and their use in disease prevention or treatment. TIFF2025530289000002.tif77170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 405,720, filed September 12, 2022, the complete disclosure of which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] background The latest developments in cell therapy and gene therapy are revolutionizing approaches to disease treatment. Gene therapy can be used to correct genetic disorders by editing the genome, for example, through the use of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) gene editing sequences. Gene therapy can also address genetic disorders by introducing new corrective genetic information, for example, through the use of clinical-grade viruses. Cell therapy can be applied to reprogram cells to direct the body's natural complex functionality toward new targets. This opens up a vast range of possible therapeutic modalities that were previously unavailable using older paradigms. Both cell therapy and gene therapy rely on the successful delivery of therapeutic gene payloads to precisely targeted cells or tissues. Many of these therapies also require the delivered genetic information to be directly integrated into the genome, allowing for long-term, heritable, and stable expression of the gene.

[0003] Retroviruses similar to the human immunodeficiency virus (HIV) are well suited to delivering and integrating genetic payloads relevant to cell and gene therapy. Cancer cell lines can be utilized as hosts for producing these retroviruses by transfecting them with multiple plasmids or vectors. Transfer vectors encode the retroviral genome to be produced by the host cell. Thus, transfer vectors contain the genetic payload, e.g., a therapeutic gene or gene circuit, to be integrated into the genome of the cell targeted by the retrovirus. Packaging plasmids encode the viral Gag-Pol proteins, which enable both retroviral production within the host cell and integration of the genetic payload into the host cell genome. Envelope plasmids encode binding proteins that enable the retrovirus to target the host cell and initiate the viral entry process.

[0004] Once a cell line produces these retroviruses, it can harness the virus to introduce new genetic material into the cell. Envelope proteins direct the virus to surface receptors on the host cell and allow the two to bind to each other, initiating the viral entry process. Once bound, proteins encoded on the transfer plasmid fuse the cell and virus together, releasing the viral contents into the cell's cytoplasm. In a complex series of steps, the viral genome is reverse-transcribed from RNA to DNA and transported into the nucleus, where viral integrase semi-randomly integrates the viral DNA into the genome. The introduced payload can then divide with the cell and undergo all other genomic processes, creating long-term changes that enable both cell and gene therapy.

[0005] However, significant challenges exist in these applications of retroviruses, greatly reducing their usefulness in more advanced cell and gene therapies, which increasingly require selective transduction of only the desired cell type, yet achieving such selectivity remains extremely difficult for several reasons.

[0006] First, viral envelope proteins often do not target specific cells of interest. The most ubiquitous envelope protein used in retroviruses is VSV-G, which binds to the LDL receptor protein. However, this family of surface proteins is absent or poorly expressed on many therapeutically important cell types. For example, natural killer (NK) cells are a type of immune cell with extremely exciting potential as therapeutics for cancer, autoimmune disorders, and diseases of aging. Unfortunately, natural killer cells do not express LDL and are therefore difficult to adapt for engineered cell therapy. The use of other envelope proteins, such as RD114 (which binds to the RDR surface protein) or BaEV (which binds to the ASCT surface protein), can at least partially overcome this problem, but often at the cost of less efficient virus production or more limited knowledge of envelope protein binding partners.

[0007] Second, viral envelope proteins lack selectivity. The binding partners of these proteins are not restricted to those expressed by specific cell types. As a result, retroviruses expressing specific envelope proteins generally cannot target specific tissues when delivered in situ. Retroviruses also typically cannot target specific cell types in a mixture when administered ex vivo. For example, currently available technology does not allow the manipulation of only T cells in peripheral blood mononuclear cell (PBMC) cultures using retroviruses as an engineering tool.

[0008] In light of these observations and results, there is a need in the art for new systems and techniques that improve the utility of retroviruses, particularly in the fields of gene therapy and cell therapy. In particular, new developments are needed to expand the range of cell types that can be manipulated by retroviruses while also making the viruses highly selective for cells or tissues of interest. The disclosure herein provides a series of solutions to address these challenges and offers related and other advantages. Summary of the Invention

[0009] Quick Overview Overall, systems, materials, and methods related to engineered retroviruses decorated with binding moieties that allow the virus to directly or indirectly bind to target cells of interest are provided herein. By utilizing certain types of binding moieties and their binding partners, such as antibodies, antibody fragments, and endogenous or engineered ligands, the provided approaches and compositions enable the design and use of retroviruses with greater specificity and flexibility than previously possible. Thus, the synthetic materials and related methods disclosed herein provide useful tools for a wide range of applications, including in the fields of gene therapy and cell engineering.

[0010] In one aspect, the present disclosure provides a retroviral vector system comprising an envelope plasmid, a packaging plasmid, and a transfer plasmid. The envelope plasmid encodes a retroviral viral membrane fusion protein. The packaging plasmid encodes a retroviral Gag-Pol protein. The transfer plasmid contains one or more genes of interest for transfer from the retrovirus to a target cell. One or both of the envelope plasmid and the packaging plasmid further encode a binding moiety. The binding moiety directly or indirectly binds to a surface feature of the target cell.

[0011] In another aspect, the present disclosure provides a retroviral packaging cell comprising any of the retroviral packaging systems disclosed herein.

[0012] In another aspect, the present disclosure provides a retrovirus comprising a viral membrane fusion protein, a viral genome, and a binding moiety. The viral genome comprises one or more genes of interest for the retrovirus to transfer into target cells. The binding moiety binds to a ligand, and the ligand comprises an antibody, an antibody mimic, a single-chain variable fragment (scFv), or a derivative or fragment thereof. The ligand binds to a surface feature of the target cell.

[0013] In another aspect, the present disclosure provides a virus-like particle comprising a binding moiety that binds to a ligand, the ligand comprising an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof, and the ligand binds to a surface feature of a target cell.

[0014] In another aspect, the present disclosure provides a method for producing a retrovirus, the method comprising transfecting a host cell with any of the retroviral vector systems disclosed herein.

[0015] In another aspect, the present disclosure provides a method for preventing or treating a disease in a subject, the method comprising administering to the subject any of the retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles disclosed herein. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a diagram of a retroviral vector system, a retroviral packaging cell, and a retrovirus, according to provided embodiments. [Figure 2]FIG. 1 is a schematic diagram of an envelope plasmid encoding a binding moiety, according to an embodiment provided. [Figure 3] 1 shows results from an example demonstrating lymphocyte transduction by provided retroviruses with CD7-targeting nanobodies as binding moieties. [Figure 4] 1 is a graph plotting the transduction of natural killer (NK) cells by the provided retroviruses or a comparative lentivirus. [Figure 5] 1 is a diagram of a universal pseudotyped retrovirus according to provided embodiments. [Figure 6] 1 is a graph plotting the transduction of Jurkat cells at various concentrations of (1) a provided retrovirus having monomeric streptavidin (mSA) as a binding moiety, and (2) a CD71 antibody conjugated to biotin. [Figure 7] 1 is a graph plotting the transduction of Jurkat cells at various concentrations of (1) a provided retrovirus having an anti-fluorescein isothiocyanate (αFITC) binding moiety, and (2) a CD71 antibody conjugated to FITC. [Figure 8] FIG. 7 is a graph plotting the magnitude of retrovirally delivered mCherry expression in the Jurkat cells of FIG. 6. [Figure 9] FIG. 8 is a graph plotting the magnitude of retrovirally delivered mCherry expression in the Jurkat cells of FIG. 7. [Figure 10] 1 is a graph plotting transduction of Jurkat cells engineered with antibodies against lymphocyte marker (CD7) or T cell marker (CD3) by provided retroviruses with a FITC single-chain variable fragment (scFv) binding moiety. [Figure 11] 1 is a graph plotting transduction of Raji cells engineered with antibodies against the B cell marker CD19 or the B cell marker CD20 by provided retroviruses bearing a FITC single-chain variable fragment (scFv) binding moiety. [Figure 12] 1 is a graph plotting transduction of primary T cells engineered with antibodies against the subtype marker CD4 or the subtype marker CD8 with provided retroviruses bearing a FITC single-chain variable fragment (scFv) binding moiety. [Figure 13] 1 is a graph plotting the specific transduction of CD4+ T cells, but not CD8+ T cells, by provided retroviruses with a FITC-binding moiety conjugated to a CD4 antibody. [Figure 14] 1 is a graph plotting the specific transduction of CD8+ T cells, but not CD4+ T cells, by provided retroviruses with a FITC-binding moiety conjugated to a CD8 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description I. Overview The present disclosure generally provides materials and methods related to retroviruses and virus-like particles engineered to have modular binding moieties on their surface. The specific binding moieties described herein decorate retroviruses and virus-like particles, enabling them to either directly bind to cells of interest or to bind to antibodies, antibody fragments, or other ligands that bind to cells. The moieties can advantageously bind to readily available, off-the-shelf antibodies, easily customized antibodies, or surface features already present on the cells being targeted.

[0018] The modular, plug-and-play approach of being able to design or select binding moieties offers several significant advantages over existing retroviral methodologies. For example, the transduction space available for retroviruses and virus-like particles can be expanded to include additional cell types that cannot be transduced by traditional lentiviral approaches. Importantly, these additional cell types include many, such as natural killer (NK) cells, which are prime candidates for targeting in developing cell and gene therapies. Binding moieties can also enable universal pseudotyping of retroviruses or virus-like particles, so that one retrovirus or virus-like particle design can be conjugated with any antibody to target any cell.

[0019] Thus, while the range of cells that can be targeted for transduction using the provided tools and techniques is broad, targeting can also advantageously be more specific than can be achieved with other retroviral approaches.By tailoring the binding moiety to recognize a specific binding partner, off-target transduction can be minimized or substantially eliminated.This reduction in undesired non-specific targeting can provide an important benefit, for example, in therapeutic applications where non-specific targeting can lead to adverse side effects.This important cellular specificity of the provided retroviruses and virus-like particles can be easily switched from one target to another due to the modular nature of the binding moiety and its activity.

[0020] The binding moieties of the provided retroviruses and virus-like particles are encoded on the packaging plasmid and / or envelope plasmid of the virus or particle. As a result, the gene encoding the binding moiety is advantageously not integrated into the host cell producing the virus or particle or targeted for transduction. Additionally, the provided systems for producing retroviruses and virus-like particles contain a minimum number of plasmids, simplifying the process and increasing efficiency. For example, the provided plasmid system for producing retroviruses can contain only three plasmids: a packaging plasmid and an envelope plasmid (at least one of which encodes the binding moiety), and a transfer plasmid carrying the genetic material to be transferred to the target cell.

[0021] The production of the provided retrovirus and virus-like particles is also advantageously simple and can be easily incorporated into standard virus production procedures.For example, a virus-producing cell such as HEK293T cell can be transfected with a transfer plasmid encoding the viral genome, an envelope plasmid encoding the viral envelope, and one or more packaging plasmids encoding the viral Gag-Pol protein.In such a system, the envelope plasmid and / or packaging plasmid can contain a binding moiety.The virus-producing cell containing these plasmids then produces functional viruses that display binding moieties on their surface.In some instances, antibodies can be incubated with purified retrovirus to conjugate the antibodies to the virus.The antibodies can then direct the retrovirus to target cells.

[0022] Figure 1 shows some exemplary aspects of the provided retroviral vector systems, retroviral packaging cells, and methods for targeting cells. Universal retroviruses can be created by engineering a packaging plasmid, or, as shown in panel (A) of the figure, an envelope plasmid, with a binding moiety. Thus, the binding moiety can comprise a binding domain fused directly to a transmembrane domain. The binding moiety can be encoded immediately upstream or downstream of the envelope or Gag-Pol protein and can be post-translationally separated from the protein by a 2A tag. Also shown in the figure, the cognate envelope protein, such as VSV-G, can be further mutated so that it no longer binds to its cognate ligand.

[0023] Panel (B) of Figure 1 shows the transfection of virus packaging cells with the engineered envelope plasmid along with the transfer and packaging plasmids. These transfected cells then produce retroviruses decorated with binding moieties, as shown in panel (C). The binding moiety can contain either a cell-binding domain (CBD, top) or an antibody / antibody fragment / ligand-binding domain (ABD, bottom). When the binding moiety contains a CBD, it directs the virus to a specific surface protein on the target cell, allowing the virus to transduce this specific cell type. When the binding moiety contains an ABD, it binds to a secondary binding agent, such as an antibody, and the combined virus and antibody together transduce the specific targeted cell type.

[0024] II. Definition Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.

[0025] As used herein, the term "retrovirus" refers to members of the Retroviridae virus family. Retroviruses have a single-stranded, diploid, positive-sense RNA genome that can be reverse-transcribed into a DNA intermediate and then integrated into the host cell genome. Viruses from the Retroviridae family are generally enveloped particles with diameters of 80 to 120 nm. Retroviral vectors or plasmids can be replication-deficient viral particles from the Retroviridae virus family. Plasmids can contain group-specific antigen (Gag) and Pol proteins, a single-stranded RNA genome, and envelope proteins. Retroviral plasmids can also contain psi elements and long terminal repeats (LTRs), which may be required for efficient packaging and reverse transcription of DNA. Retroviruses include alpharetroviruses, gammaretroviruses, and lentiviruses. Representative species of lentiviruses include human immunodeficiency virus (HIV). Representative species of gammaretroviruses include murine leukemia virus and feline leukemia virus.

[0026] Integrase-deficient retroviruses and retroviral vectors cannot integrate retroviral vector genomes into host cell genomes.Integrase-deficient retroviral vectors or plasmids can be derived from conventional retroviral vectors, and lack retroviral integrase or contain a mutant form thereof.When entering host cells, the retroviral vector genome of integrase-deficient retroviruses is reverse transcribed in the cytoplasm and delivered into the nucleus, but is not stably integrated into the host cell genome.

[0027] As used herein, the term "transduction" refers to the process by which a virus enters a host cell and delivers its RNA genome, and the expression by the host cell of the gene of interest thus delivered.

[0028] As used herein, the terms "virus-like particle" and "VLP" refer to particles that resemble viruses but do not contain viral genetic material encoding the proteins of virus-like particles, and therefore do not infect or transduce. The expression of viral structural proteins, such as envelope or capsid proteins, can result in the assembly of virus-like particles. Virus-like particles can be used to deliver proteins and / or nucleic acids to the cytoplasm of target cells.

[0029] As used herein, the term "plasmid" refers to a circular double-stranded DNA containing one or more sequences of interest, for example, a sequence encoding one or more specific proteins. In some embodiments, the plasmid may further comprise a regulatory sequence or other genetic element that is operably linked to the sequence encoding the specific protein.

[0030] As used herein, the terms "viral membrane fusion protein," "membrane fusion protein," "fusion protein," and "fusogen" refer to a polypeptide that causes or enhances the fusion of biological membranes, such as a viral envelope and a cell wall. A viral membrane fusion protein can be a transmembrane protein, or a functional fragment or derivative thereof.

[0031] As used herein, the term "cell" generally refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of an organism. A cell can originate from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, and mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum spp., and the like), and patens, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), cells from invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, mollusks, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cells do not originate from a natural organism (e.g., cells can be synthetically made, sometimes called artificial cells).

[0032] As used herein, the term "variant" in the context of the polypeptides described herein refers to polypeptides that have a high degree of structural similarity with each other, with structural differences resulting from differences in the polynucleotides encoding the polypeptide variants. Polypeptide variants can have amino acid sequences that are at least 80% similar (% identity) to each other, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar (% identity) to each other. Polypeptide variants can have the same biological function as each other. For example, if the polypeptide variants are enzymes, the polypeptide variants can each catalyze the same reaction. Alternatively, variants of an original polypeptide can be specifically constructed or selected to lack the biological activity or function of the original polypeptide.

[0033] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family, or a polypeptide comprising an immunoglobulin fragment, capable of noncovalently, reversibly, and specifically binding to an epitope of a corresponding antigen. The term includes, but is not limited to, polyclonal or monoclonal antibodies of the isotype classes IgA, IgD, IgE, IgG, and IgM, derived from human or other mammalian cells, including naturally occurring or genetically engineered forms, such as humanized antibodies, human antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, grafted antibodies, and in vitro-generated antibodies. The term encompasses proteins containing immunoglobulin moieties (e.g., chimeric or bispecific antibodies or single-chain Fvs (scFvs)), and conjugates, including, but not limited to, fragments such as Fab, F(ab'), Fv, scFv, Fd, dAb, and other compositions.

[0034] As used herein, the terms "single-chain variable fragment," "single-chain Fv," and "scFv" refer to antibodies in which the heavy and light chain variable domains of a traditional two-chain antibody are linked to form a single chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.

[0035] As used herein, the term "nanobody" or "single domain antibody" refers to an antibody fragment that is composed of a single monomeric variable antibody domain, has a molecular weight of less than 20 kDa, and is capable of selectively binding to a specific antigen.

[0036] As used herein, the term "epitope" refers to a localized site on an antigen that an antibody recognizes and binds to. A protein epitope can comprise a small number of amino acids or a portion of a small number of amino acids, for example, 5, 6, or more, or 20 or more amino acids or a portion of those amino acids. An epitope can also comprise non-protein components, such as nucleic acids (e.g., RNA or DNA), carbohydrates, lipids, or a combination thereof. An epitope can be a three-dimensional moiety. Thus, for example, if the target is a protein target, the epitope can comprise consecutive amino acids or amino acids from different parts of the protein that are adjacent due to protein folding (e.g., a discontinuous epitope). The same applies to other types of target molecules, such as DNA and chromatin, that form three-dimensional structures.

[0037] As used herein, the terms "specifically (or selectively) bind," or, when referring to antibody interactions, "specifically (or selectively) immunoreactive with," refer to a binding reaction between two molecules that is at least twice background, and more typically 10-100 times greater than background molecular association, under physiological conditions. When one or more detectable binding agents are used that are proteins, specific binding determines the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, a specialized antibody binds to a specific protein sequence, thereby identifying its presence.

[0038] Specific binding to an antibody under such conditions requires that the antibody be selected for its specificity to a particular protein. For example, antibodies raised against a particular protein, polymorphic variants, alleles, orthologs, and conservatively modified variants or splice variants, or portions thereof, can be selected to obtain only polyclonal antibodies that are specifically immunoreactive with the protein of interest and not with other proteins. This selection can be achieved by subtracting antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with a protein (see, for example, Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods for determining whether two molecules specifically interact are disclosed herein, and methods for determining binding affinity and specificity are well known in the art (see, e.g., Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Friefelder, "Physical Biochemistry: Applications to biochemistry and molecular biology" (WH Freeman and Co. 1976)).

[0039] As used herein, the terms "switch receptor" and "chimeric switch receptor" refer to a molecule designed to switch a negative signaling signal to a positive signal. The switch receptor can be a chimeric protein comprising a first protein or a fragment thereof associated with a negative signal and a second protein or a fragment thereof associated with a positive signal. Examples of proteins associated with negative signals include, but are not limited to, CTLA-4, PD-1, BTLA, TIM-3, etc. Examples of proteins associated with positive signals include, but are not limited to, CD28, ICOS, 4-1BB, TGFβR, etc.

[0040] As used herein, the term "selectable marker" refers to a gene that encodes a protein that allows cells expressing the gene to be identified and / or separated from other cells in a population. Selectable markers include, but are not limited to, genes encoding drug resistance, fluorescence, and genes essential for growth under restrictive conditions.

[0041] As used herein, the term "subject" refers to a vertebrate, preferably a mammal. Mammalian subjects for which the provided compositions are suitable include, but are not limited to, mice, rats, apes, humans, livestock, sport animals, and pets. In some embodiments, the subject is a human. In some embodiments, the subject is a male. In some embodiments, the subject is a female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is over 10 years old, for example, over 20 years old, over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, or over 80 years old. In some embodiments, the subject is under 80 years old, for example, under 70 years old, under 60 years old, under 50 years old, under 40 years old, under 30 years old, under 20 years old, or under 10 years old.

[0042] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, or subcutaneous administration, intrathecal administration, or implantation of a sustained release device, e.g., a mini-osmotic pump, into a subject.

[0043] As used herein, the terms "treat," "treating," and "treatment" refer to a procedure that results in any indication of successful elimination or amelioration of an injury, condition, state, or symptom (e.g., pain), including any objective or subjective parameter, such as relief; remission; a decrease in symptoms or making the symptom, injury, medical condition, or condition more tolerable to the patient; a decrease in the frequency or duration of the symptom or condition; or, in some circumstances, prevention of the onset of the symptom. Treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0044] As used herein, the terms "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of an active agent by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the subject. Non-limiting examples of pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, etc. Those skilled in the art will recognize that other pharmaceutically acceptable excipients and carriers are useful in the present disclosure.

[0045] As used herein, the term "therapeutically effective amount" refers to the amount or dosage of a compound, composition or preparation that produces the therapeutic effect for which it is administered.The exact amount or dosage depends on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0046] As used herein, the term "vaccine" refers to a composition comprising at least one antigen or immunogen in a pharmaceutically acceptable carrier, or a nucleic acid molecule encoding at least one antigen or immunogen, that is useful for inducing an immune response to the antigen or immunogen in a subject for the purpose of improving immunity to disease and / or infection in the subject.

[0047] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polymer" optionally includes combinations of two or more polymers, and the like.

[0048] As used herein, the term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0049] As used herein, the terms "including," "comprising," "having," "containing," and variations thereof are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those expressly recited. As used herein, the phrase "consisting of" is exclusive and excludes any element, step, or ingredient not expressly specified. As used herein, the phrase "consisting essentially of" limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel properties of the disclosed feature.

[0050] III. Retroviral Vector Systems In one aspect of the present disclosure, a retroviral vector system is provided. The vector system includes a plasmid that can be used to transform retroviral packaging cells, allowing the cells to produce retrovirus according to the contents of the vector system. Thus, the disclosed retroviral vector system can provide many of the surprising advantages discussed herein, particularly when used in therapeutic applications such as gene therapy or cell therapy. For example, the retroviral vector system can be used as a tool to generate retroviruses engineered to specifically and / or selectively transduce one or more specific cell types or populations. In certain aspects, the engineered retroviruses generated using the retroviral vector system can also advantageously have easily switchable cell specificity. This switchable specificity can be achieved, for example, by using a simple procedure to change the binding moiety of the retrovirus or the secondary binding agent, e.g., an antibody, to which the retroviral binding moiety is conjugated.

[0051] The provided retroviral vector systems comprise one or more envelope plasmids, one or more packaging plasmids, and one or more transfer plasmids. An advantage of the provided vector systems is that a small number of plasmids can be effective in transfecting virus packaging cells to enable the cells to produce functional retroviruses having the binding moieties disclosed herein. In some embodiments, the retroviral vector system comprises only one envelope plasmid. In some embodiments, the retroviral vector system comprises only one packaging plasmid. In some embodiments, the retroviral vector system comprises only one transfer plasmid. In some embodiments, the retroviral vector system comprises only one envelope plasmid and one packaging plasmid. In some embodiments, the retroviral packaging system comprises only one envelope plasmid and one transfer plasmid. In some embodiments, the retroviral vector system comprises only one packaging plasmid and one transfer plasmid. In some embodiments, the retroviral vector system comprises only one envelope plasmid, one packaging plasmid, and one transfer plasmid.

[0052] The envelope plasmid of the provided retroviral vector system generally encodes the viral membrane fusion protein of the retrovirus produced by the viral packaging cell transfected with the vector system. The packaging plasmid of the retroviral vector system generally encodes the Gag-Pol protein of the retrovirus. The transfer plasmid of the retroviral vector system generally encodes one or more genes of interest for transfer from the retrovirus to target cells.

[0053] At least one plasmid in the provided retroviral vector system encodes a binding moiety that directly or indirectly binds to a surface feature of a target cell. Encoding a binding moiety in one of the vector system's plasmids advantageously ensures that all retroviruses produced using the system contain a binding moiety. In some embodiments, only one plasmid in the retroviral vector system encodes a binding moiety. In some embodiments, two or more different plasmids in the retroviral vector system encode a binding moiety. In some embodiments, each different plasmid in the retroviral vector system encodes a binding moiety.

[0054] For certain applications, it may be desirable to encode the junction moiety only in one or both of the envelope plasmid and the packaging plasmid, but not in the transfer plasmid. In this way, the junction moiety will not be integrated into the genome of the host cell targeted by the retrovirus produced using the vector system. In some embodiments, only one or more envelope plasmids encode the junction moiety. In some embodiments, only one or more packaging plasmids encode the junction moiety. In some embodiments, the envelope plasmid and the packaging plasmid each encode the junction moiety, but the transfer plasmid does not.

[0055] The binding moiety of the provided retroviral vector systems generally comprises an extramembrane domain, where the extramembrane domain is a binding domain that recognizes a surface feature of a target cell or a secondary binding partner that recognizes a surface feature of a target cell. In some embodiments, the binding moiety comprises a transmembrane domain that positions the binding moiety in the retroviral envelope. In some embodiments, the extramembrane domain of the binding moiety is fused to the transmembrane domain of another viral envelope protein.

[0056] The binding moiety can be encoded immediately upstream of the envelope protein on the envelope plasmid of the provided retroviral vector system. The binding moiety can be encoded immediately downstream of the envelope protein on the envelope plasmid (Figure 2). The binding moiety can be encoded immediately upstream of the Gag-Pol protein on the packaging plasmid of the retroviral vector system. The binding moiety can be encoded immediately downstream of the Gag-Pol protein on the packaging plasmid. In some embodiments, the binding moiety is post-translationally separated from adjacent proteins of the plasmid, for example, by a 2A tag.

[0057] In some embodiments, the binding moiety of the provided retroviral vector system is designed or selected to bind to a ligand, where the ligand is designed or selected to bind to a target cell, e.g., to a surface feature of the target cell (Figure 1). In some embodiments, the ligand to which the binding moiety binds includes an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof. In such embodiments, the binding moiety can be described as comprising an antibody binding domain (ABD).

[0058] In some embodiments, the ligand is a conjugated derivative of an antibody, antibody mimetic, or scFv, where the ligand is conjugated to a small molecule recognized by the binding moiety (Figure 5). Using this approach, retroviral vector systems can be designed so that they can be used to generate universal pseudotyped retroviruses. Each of these universal pseudotyped retroviruses contains a binding moiety that binds to a specific small molecule, and any conjugate between the ligand and the small molecule can be easily created so that the ligand is compatible with the binding moiety that has affinity for the specific small molecule of the conjugate. In this way, the single universal design of the provided retrovirus, for example, a retrovirus generated using the provided retroviral vector system, can specifically bind to a specific cell type when a ligand-small molecule conjugate specific to that cell type is applied.

[0059] In some embodiments, the binding moiety comprises a fluorescein isothiocyanate (FITC) binding domain, and the ligand is conjugated to FITC. For example, the binding moiety can comprise an anti-fluorescein scFv or a fluorescein-conjugated anticalin. The ligand recognized by this binding moiety can be, for example, an FITC-conjugated antibody. Due to the availability of FITC-conjugated antibodies specific for a variety of different cell-specific antigens, a single design of a universal pseudotyped retrovirus with an FITC-binding moiety can specifically transduce a variety of cell types.

[0060] In some embodiments, the binding moiety comprises a biotin-binding domain, and the ligand is biotin-conjugated. For example, the binding moiety can comprise an anti-biotin scFv, a biotin-binding anticalin, or an avidin family protein, such as avidin or streptavidin. The ligand recognized by this binding moiety can be, for example, a biotin-conjugated antibody. Due to the availability of biotin-conjugated antibodies specific for a variety of different cell-specific antigens, a single design of universal pseudotyped retrovirus with a biotin-binding moiety can specifically transduce a variety of cell types.

[0061] The ligand recognized by the antibody binding domain of the provided binding moiety can itself bind to a wide variety of surface features of target cells.Many pre-made antibodies suitable for use in the provided materials and methods are available, where the available antibodies have different specificities and selectivities for various antigens and epitopes.Additional antibodies and antibody conjugates can be developed for binding to additional target cell surface features.For example, but not limited to, a ligand can be used to indirectly bind the provided binding moiety to one or more of CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.

[0062] In some embodiments, the binding moiety of the provided retroviral vector system is designed or selected to directly bind to a target cell, e.g., to a surface feature of the target cell (Figure 1). In such embodiments, the binding moiety can be described as comprising a cell-binding domain (CBD). In some embodiments, the cell-binding domain comprises an antibody, an antibody mimetic, an scFv, a nanobody, another ligand, or a derivative or fragment thereof. Similar to binding moieties comprising antibody-binding domains, binding moieties comprising cell-binding domains can also bind to a wide variety of surface features of target cells. For example, but not limited to, the cell-binding domain can be designed or selected to allow the provided binding moiety to bind to one or more of CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.

[0063] A wide variety of cell types can be targeted by the provided retroviral vector system.In some embodiments, target cells are immune cells, including any cells involved in immune response.For example, targeting immune cells, such as optionally allogeneic natural NK cells, iPSC-derived NK cells, and / or macrophage cells, can greatly facilitate the treatment of solid tumors while avoiding side effects.In this way, the provided retroviral vector system can mediate the precise activation of immunity at precise location (for example, the presence of local tumor signal) and specific time point.

[0064] In some embodiments, target cells include granulocytes, such as basophils, eosinophils, and neutrophils; mast cells; monocytes that can develop into macrophages; antigen-presenting cells, such as dendritic cells; and lymphocytes, such as natural killer cells (NK cells), B cells, and T cells. In some embodiments, target cells are immune effector cells. Immune effector cells are immune cells that can perform a specific function in response to a stimulus. In some embodiments, target cells are immune effector cells that can induce cell death. In some embodiments, target cells are lymphocytes. In some embodiments, the lymphocytes are NK cells. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are activated T cells. T cells include both naive and memory cells (e.g., central memory or T CM , Effector Memory or T EM , and effector memory RA or T EMRA ), effector cells (e.g., cytotoxic T cells or CTL or Tc cells), helper cells (e.g., Th1, Th2, Th3, Th9, Th7, TfH), regulatory cells (e.g., Treg and Trl cells), natural killer T cells (NKT cells), tumor-infiltrating lymphocytes (TIL), lymphocyte-activated killer cells (LAK), αβ T cells, γδ T cells, and similar unique classes of T cell lineages.

[0065] T cells can be divided into two broad categories: CD8+ T cells and CD4+ T cells, based on which proteins are present on the cell surface. T cells can perform multiple functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are called cytotoxic T cells or cytotoxic T lymphocytes (CTLs). CD4+ T cells can be subdivided into four subsets: Th1, Th2, Th17, and Treg. "Th" refers to "T helper cells," although additional subsets may exist. Th1 cells can orchestrate immune responses to intracellular microorganisms, particularly bacteria. They can produce and secrete molecules that alert and activate other immune cells, such as macrophages, which ingest bacteria. Th2 cells are involved in orchestrating immune responses to extracellular pathogens, such as helminths (parasites), by alerting B cells, granulocytes, and mast cells. Th17 cells can produce interleukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells, and are important for recruiting neutrophils.

[0066] Retroviral vector systems can be engineered to target cell types that mediate cell differentiation and programming. This can enable site-specific differentiation of cells and tissues to repair or regenerate damaged or aging bodies. In some embodiments, the target cell is a stem cell. The target cell can be, for example, an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), an adult stem cell, or a mesenchymal stem cell (MSC). In some embodiments, the target cell is a progenitor cell. The target cell can be, for example, a neural progenitor cell, a skeletal progenitor cell, a muscle progenitor cell, adipose progenitor cell, a cardiac progenitor cell, a chondrocyte, or a pancreatic progenitor cell.

[0067] In some embodiments, the provided retroviral vector systems are designed to generate retroviruses that target cells of one or more specific tissues. These embodiments can be particularly useful when the retroviral vector systems are used to generate retroviruses for in situ manipulation or treatment of those specific tissues. In some embodiments, the target cells include muscle cells. In some embodiments, the target cells include neural cells. In some embodiments, the target cells include pancreatic cells, such as pancreatic islets.

[0068] In some embodiments, provided retroviral vector system is designed to produce retroviruses that target two or more different cell types, and each of the retroviral vector systems has a different payload that is delivered to each of the different cell types.For example, the retroviral vector system can comprise a FITC binding domain that recognizes a FITC-conjugated antibody, where the FITC-conjugated antibody targets a first antigen for delivery of a first payload.The same retroviral vector system can further comprise a biotin binding domain that recognizes a biotin-conjugated antibody, where the biotin-conjugated antibody targets a second antigen for delivery of a second payload.

[0069] The provided retroviral vector system can be used to deliver any gene-encoded material. The system and the retrovirus it produces can be used to deliver, for example, human genes, chimeric and engineered genes, gene editors, and genes from other species. In some embodiments, the vector system transfer plasmid gene encodes a receptor, including, but not limited to, a chimeric antigen receptor (CAR) (including first-, second-, third-, or fourth-generation receptors), a switch receptor, a MIMIC receptor, or modified versions thereof. In some embodiments, the transfer vector genes are selected from the group consisting of CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and Natronobacterium gregorii (Natronobacterium gregorii). gregoryi Argonaute (NgAgo); adenosine deaminase acting on RNA (ADAR); one or more nucleases, such as CIRT, PUF, homing endonuclease, or functional fragments, derivatives, or variants thereof. In some embodiments, the transfer vector gene comprises one or more reporter genes or selectable markers, such as fluorescent proteins, antibiotic resistance genes, or derivatives or fragments thereof.

[0070] In some embodiments, provided transfer plasmid comprises more than one gene in one or more polycistronic elements.In some embodiments, retroviral vector system can be used to create retrovirus, for example, using IRES or 2A tag, to transduce host cell to express more than one gene of polycistronic element.In some embodiments, transfer plasmid comprises one or more regulatory elements, such as promoter, intron, enhancer, post-transcriptional element or post-translational element, that can regulate transduced gene expression.

[0071] In some embodiments, the viral membrane fusion protein of the provided retroviral vector system envelope plasmid is the vesicular stomatitis virus G (VSV-G) protein or an engineered variant thereof. In some embodiments, the viral membrane fusion protein is the membrane fusion protein derived from feline endogenous virus RD114 or an engineered variant thereof. In some embodiments, the viral membrane fusion protein is the membrane fusion protein derived from baboon endogenous virus BaEV or an engineered variant thereof.

[0072] In some embodiments, the viral membrane fusion protein is an engineered variant that does not bind to the cognate binding partner of the corresponding wild-type viral membrane fusion protein.This can be particularly advantageous in applications where it is important to ensure that the retrovirus produced using a retroviral vector system relies on the binding part of the vector system for host cell targeting and transduction.In some embodiments, the engineered variant comprises one or more, for example, two or more point mutations in the sequence of the wild-type viral membrane fusion protein, so that the protein loses its cognate binding ability.In some embodiments, the engineered variant is a truncated mutant of the wild-type viral membrane fusion protein, so that the protein loses its cognate binding ability.

[0073] In some embodiments, the integrase enzyme of the provided retroviral vector systems is mutated such that the vector systems can be used to generate integrase-deficient retroviruses, which can be particularly advantageous for applications seeking to deliver genetic information to target cells without stable integration of that information into the target cell genome.

[0074] IV. Retroviral Packaging Cells In another aspect of the present disclosure, retroviral packaging cells are provided. Retroviral packaging cells are cells that have been transformed with any of the provided retroviral vector systems, such as those described in Section III. Thus, retroviral packaging cells can provide many of the surprising advantages discussed herein, particularly when used in therapeutic applications such as gene therapy or cell therapy. For example, retroviral packaging cells can be used as a tool to generate retroviruses engineered to specifically and / or selectively transduce one or more particular cell types or populations. In certain aspects, engineered retroviruses produced using retroviral packaging cells can also advantageously have easily switchable target cell specificity. This switchable specificity can be achieved, for example, by using a facile procedure to alter the binding moiety of the retrovirus or the secondary binding agent, e.g., an antibody, to which the retroviral binding moiety is conjugated.

[0075] In another aspect, a population of retroviral packaging cells is provided. In some embodiments, each host cell of the population independently comprises a retroviral vector system as disclosed herein. Also provided is a cell culture comprising a population of cells as described herein. Methods for culturing and producing many cells, including cells of bacterial (e.g., E. coli and other bacterial strains), animal (particularly mammalian), and archaeal origin, are available in the art. See, for example, Sambrook, supra; Ausubel, ed. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, and Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3 rd Ed., Wiley-Liss, New York, and the references cited therein: Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, 4th Ed. W.H. Freeman and Company; and Ricciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024.

[0076] V. Retroviruses In another aspect of the present disclosure, retrovirus is provided.In some embodiments, retrovirus can be produced using any of provided retrovirus vector systems and / or retrovirus packaging systems.Therefore, retrovirus can provide the surprising advantages discussed herein, including wide applicability, high specificity and selectivity, switchable targeting and universal pseudotyping.

[0077] The provided retroviruses comprise a viral membrane fusion protein. The viral membrane fusion protein can be any of those disclosed herein with respect to the retroviral vector system. The provided retroviruses further comprise a viral genome comprising one or more genes of interest for transfer from the retrovirus to a target cell. The genes of interest and the target cell can be any of those disclosed herein with respect to the retroviral vector system. The provided retroviruses further comprise a binding moiety, which can be any of those disclosed herein with respect to the retroviral vector system. In some embodiments, the binding moiety comprises an antibody binding domain (ABD) of any of the types disclosed herein. For example, the binding moiety of the provided retroviruses can comprise a ligand that is an antibody, antibody mimetic, single-chain variable fragment (scFv), or a derivative or fragment thereof, wherein the ligand binds to a surface feature of the target cell.

[0078] VI. Virus-like particles In another aspect of the present disclosure, virus-like particles are provided.Virus-like particles comprise many of the same advantages as provided retroviruses, including wide applicability, high specificity and selectivity, switchable targeting and universal pseudotyping.Different from provided retroviruses, virus-like particles do not comprise viral genome, and therefore cannot be used to integrate delivered genetic material into the genome of target cell.Therefore, virus-like particles offer an attractive alternative to retroviruses when such integration is undesirable.

[0079] The provided virus-like particles comprise a binding moiety, which can be any of those disclosed herein for retroviral vector systems. In some embodiments, the binding moiety comprises any of the antibody binding domains (ABDs) of the types disclosed herein. For example, the binding moiety of the provided retrovirus can comprise a ligand that is an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof, wherein the ligand binds to a surface feature of a target cell.

[0080] VII. Methods for Preventing or Treating Disease In another aspect of the present disclosure, methods for preventing or treating disease are provided. The methods include administering to a subject any of the retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles disclosed herein, e.g., in Sections III, IV, V, and VI. Pharmaceutical compositions containing the retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles described herein, e.g., in Section IX, can be administered for prophylactic and / or therapeutic treatment. In therapeutic applications, these compositions can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially inhibit the symptoms of the disease or condition, or to cure, relieve, improve, or ameliorate the condition. Amounts effective for this use can vary based on the severity and course of the disease or condition, previous treatments, the subject's health, weight, and response to drugs, and the judgment of the treating physician.

[0081] The retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administering the compositions can vary. For example, pharmaceutical compositions can be used as prophylactics and administered continuously to subjects with a condition or tendency to a disease to prevent the onset of the disease or condition. The pharmaceutical compositions can be administered to a subject during the onset of symptoms or as soon as possible after onset. Administration can begin within the first 48 hours, the first 24 hours, the first 6 hours, or within 3 hours of the onset of symptoms. The initial administration can be via any practical route, such as by any route described herein, using any formulation described herein. The compositions can be administered as soon as practicable after the onset of a disease or condition is detected or suspected, for the length of time necessary to treat the disease, such as from about 1 month to about 3 months. The length of treatment can vary for each subject.

[0082] A wide variety of diseases can be prevented or treated using the provided methods, which are suitable for a wide range of cell or gene therapy applications, for example, to treat hematological or solid cancers, viral infections, bacterial infections, genetic diseases, wound healing, autoimmunity, regenerative medicine, CNS diseases, and anti-aging.

[0083] In some embodiments, the disease that is prevented or treated is hereditary disorder.The disease that is suitable for being treated by provided method includes but is not limited to X-linked severe combined immunodeficiency syndrome, sickle cell anemia, thalassemia, hemophilia, neoplasia, cancer, age-related macular degeneration, schizophrenia, triplet repeat disease, fragile X syndrome, prion-related disorder, amyotrophic lateral sclerosis, drug addiction, autism, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood and coagulation disease or disorder, inflammation, facioscapulohumeral muscular dystrophy, retinitis pigmentosa, Leber's congenital amaurosis, glaucoma, immune-related disease or disorder, metabolic disease and disorder, liver disease and disorder, kidney disease and disorder, musculoskeletal disease and disorder, nerve and neuron disease and disorder, cardiovascular disease and disorder, lung disease and disorder and eye disease and disorder.

[0084] In some embodiments, the disease to be prevented or treated is cancer. Non-limiting examples of cancers that can be treated with the provided retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adrenocortical carcinoma, adult T Cell leukemia, aggressive NK cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, Cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, carcinoma of the penis, carcinoma of unknown primary site, carcinosarcoma, Castleman's disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct carcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell Lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, nasal neuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, inclusion fetal malformation, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma,Gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, Islet cell carcinoma, Islet cell tumor, Juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, Kidney cancer, Krackin's tumor, Krukenberg's tumor, Laryngeal cancer, Laryngeal cancer, Laryngeal lentigo maligna melanoma, Leukemia, Leukemia, Cancer of the lip and oral cavity, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant fibrous histiocytoma, Malignant fibrous histiocytoma of bone, Malignant glioma, Malignant mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant Triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myeloid leukemia, myeloid sarcoma, myeloproliferative disorder, myxoma, nasal cavity cancer, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neuroblastoma fibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor,Pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, polyblastoma, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract cancer related to the NUT gene on chromosome 15 Cancer, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, Somatostatinoma, sooty warts, spinal cord tumor, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, capsular cell carcinoma These include: cysts, throat cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof.

[0085] In some embodiments, the disease to be prevented or treated is a cancerous tumor. The cancerous tumor can be a solid cancerous tumor or a liquid cancerous tumor. The liquid cancerous tumor can be, for example, lymphoma or leukemia. Tumors treated with the methods disclosed herein can result in stabilized tumor growth (e.g., one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20% and / or do not metastasize). In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years or more. In some embodiments, the size of the tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the tumor is completely eliminated or reduced below the level of detection. In some embodiments, the subject remains tumor-free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or more after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more following treatment.

[0086] Those skilled in the art will also recognize that the provided systems and compositions can be co-administered with other therapeutic agents for cancer treatment. Anticancer agents suitable for combination therapy include, but are not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, interferons, radiopharmaceuticals, peptides with antitumor activity such as TNF-α, pharmaceutically acceptable salts thereof; derivatives thereof, prodrugs thereof, and combinations thereof. For example, pharmaceutical compositions comprising the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles can be administered to patients before, during, or after chemotherapy, or before, during, or after the administration of an anticancer agent or a combination of anticancer agents.

[0087] In some embodiments, treatment with the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles results in stable disease, partial remission, or complete remission in the subject (e.g., the methods described herein include administering to a subject a dose of a provided retroviral vector system, retroviral packaging cells, retrovirus, and / or virus-like particle that kills or otherwise slows the growth or progression of cancer cells, resulting in stable disease or partial or complete remission of cancer in the subject). In some embodiments, treatment with the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles results in reduced metastasis of cancer in the subject (e.g., the methods described herein include administering to a subject a dose of a provided retroviral vector system, retroviral packaging cells, retrovirus, and / or virus-like particle that reduces metastasis of cancer in the subject). In some embodiments, treatment with a provided retroviral vector system, retroviral packaging cells, retrovirus, and / or virus-like particle results in a reduction in tumor volume, size, or growth in the subject (e.g., the methods described herein include administering to a subject a dose of a provided retroviral vector system, retroviral packaging cells, retrovirus, and / or virus-like particle that reduces tumor volume, size, or growth in the subject). In some embodiments, treatment with a provided retroviral vector system, retroviral packaging cells, retrovirus, and / or virus-like particle results in an increase in the responsiveness of the cancer to a subsequently administered anti-cancer agent (e.g., the methods described herein include administering to a subject a dose of a provided retroviral vector system, retroviral packaging cells, retrovirus, and / or virus-like particle that increases the responsiveness of the cancer to a subsequently administered anti-cancer agent).

[0088] In some embodiments, the disease to be prevented or treated is an infectious disease. The infectious disease can be, for example, a viral infectious disease. The infectious disease can be, for example, a bacterial infectious disease. In the case of bacterial infections, the innate immune system must recognize specific markers of microorganisms to eliminate the pathogen. These pathogen-associated molecular patterns are recognized by various receptors, most notably TLRs, which are common to all immune cells and act to activate immune pathways that activate their bactericidal capabilities. In the case of macrophages, recognition of microbial pathogens through TLRs activates their unique ability to engulf the bacteria within themselves and destroy the pathogen by acidification. However, bacteria have a mechanism to evade macrophages by hiding the molecules that trigger this activation. For example, during the implantation of biomedical devices such as catheters and pacemakers, bacteria form dense biofilms around themselves to avoid recognition, causing massive infection problems and preventing life-saving technologies from entering the clinic. The provided retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles can "rewire" these TLRs to recognize components of the biofilm itself as opposed to bacteria, thus activating macrophages as an evasion mechanism to destroy the infection and making these devices safer to implant.

[0089] In some embodiments, the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles are administered to a subject once, twice, three times, four times, or five times over the course of treatment. Subsequent administrations of the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles can be performed at defined time intervals, such as days, weeks, or months apart. In some cases, if tumors or cancerous cells reappear, continue to grow, or are otherwise not completely treated after the initial administration of the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles, the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles are subsequently administered. In some cases, if a subject does not have a complete response to the initial treatment, but experiences a partial response, stable response, or progressive disease, the provided retroviral vector systems, retroviral packaging cells, retroviruses, and / or virus-like particles are subsequently administered again.

[0090] In some embodiments, the provided method further comprises obtaining a test sample from a subject. The test sample can include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof. In some embodiments, the provided method further comprises determining the level of one or more biomarkers in the obtained test sample. The step of determining the presence or level of a biomarker can be used, for example, to determine the response to treatment or to select a suitable composition for preventing or treating a disease.

[0091] In some embodiments, the provided method further comprises comparing the determined level of one or more biomarkers in the obtained test sample with the level of one or more biomarkers in a reference sample.The reference sample can be, for example, obtained from a subject, and the reference sample is obtained before obtaining the test sample, for example, before administering a therapeutically effective amount of the provided material to the subject.In this way, the reference sample can provide information about the baseline level of biomarkers in the sample before treatment, and the test sample can provide information about the level of biomarkers after treatment.

[0092] Alternatively, the reference sample can be obtained, for example, from a different subject, for example, from a subject who has not been treated according to the provided method.In this way, the reference sample can provide information about the baseline level of biomarkers without treatment, and the test sample can provide information about the level of biomarkers with treatment.The reference sample can also be obtained, for example, from a population of subjects, for example, from a population of subjects who have not been treated according to the provided method.In this way, the reference sample can provide population average information about the baseline level of biomarkers without treatment, and the test sample can provide information about the level of biomarkers with treatment.

[0093] A reference sample can also be obtained from an individual or a population of individuals after treatment has been provided according to the provided method, and can serve as, for example, a positive control sample. In some embodiments, the reference sample is obtained from normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue.

[0094] Depending on the biomarker, an increase or decrease compared to a normal control or reference sample can indicate the presence of disease or a response to treatment for the disease. In some embodiments, when the biomarker level is at least 1.1 times higher than the negative control, for example, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times higher, the increase in the level of the biomarker in the test sample, and therefore the presence of a disease, for example, an infectious disease or cancer, an increased risk of disease, or a response to treatment, is determined. In other embodiments, a decreased level of the biomarker in the test sample, and thus the presence of disease, an increased risk of disease, or a response to treatment, is determined if the biomarker level is at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold lower compared to the negative control.

[0095] Biomarker level can be detected by any method known in the art, including the use of biomarker-specific antibody.Exemplary methods include but are not limited to polymerase chain reaction (PCR), Western blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assay, for example, using Luminex or fluorescent microbeads.In some examples, nucleic acid sequencing is used.

[0096] In certain embodiments, the presence of a decrease or increase in the level of one or more biomarkers is indicated by a detectable signal, such as blot, fluorescence, chemiluminescence, color, or radioactivity in an immunoassay or PCR reaction, such as quantitative PCR.This detectable signal can be compared with the signal from a reference sample or a threshold value.

[0097] In some embodiments, the results of determining the biomarker level are recorded in a tangible medium. For example, the results of a diagnostic assay, such as the observation of the presence or decrease or increase of one or more biomarkers, and the diagnosis of whether there is an increased risk or presence of a disease, such as an infectious disease or cancer, or whether the subject is responding to treatment, can be recorded on paper or electronic media, such as an audio tape, a computer disk, a CD-ROM, or a flash drive.

[0098] In some embodiments, the methods provided further include providing the results of the diagnosis and / or treatment to the subject.

[0099] VIII. Methods for Inducing an Immune Response In another aspect, the present disclosure provides various methods for inducing an immune response in a subject. The methods generally include administering to a subject any of the retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles disclosed herein, for example, in Sections III, IV, V, and VI. For example, an immunogenic composition can be formed that includes any of the provided retroviral vector systems, retroviral packaging cells, retroviruses, or virus-like particles. The immunogenic composition can be a vaccine, and administering the immunogenic composition includes vaccinating the subject.

[0100] The disclosed immunogenic compositions can be administered using the provided methods as a single dose or multiple doses, for example, as two doses administered at intervals of about 1 week, 2 weeks, 3 weeks, 1 month, about 2 months, about 3 months, about 6 months, or about 12 months.Other suitable dosing schedules can be determined by a physician.In some embodiments, additional compounds or drugs can be co-administered to a subject.Such compounds or drugs can be co-administered, for example, to alleviate the signs or symptoms of the disease being treated or to reduce the side effects caused by the induction of an immune response.

[0101] IX. Pharmaceutical Compositions In another aspect, pharmaceutical compositions are provided.Provided pharmaceutical compositions comprise one or more, for example, two or more, of the retroviral vector system, retroviral packaging cell, retrovirus or virus-like particle disclosed herein, for example, in Section III, IV, V and VI.Provided pharmaceutical compositions can, for example, better enable retrovirus or virus-like particle disclosed herein to deliver gene payload to the subject in need thereof in situ.

[0102] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises one or more diluents, adjuvants, or carriers in a formulation suitable for administration, for example, to a mammal. Suitable diluents, adjuvants, or carriers may include, for example, lipids, such as liposomes, for example, liposomal dendrimers; liquids, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.; acacia gum; gelatin; starch paste; talc; keratin; colloidal silica; urea, etc. Additional examples of suitable diluents include distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. The pharmaceutical composition may also contain additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and surfactants. In addition, auxiliary agents, thickeners, lubricants, and coloring agents may alternatively or additionally be used.The pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

[0103] The provided pharmaceutical compositions can also include any of a variety of stabilizers, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with a variety of well-known compounds that enhance the polypeptide's in vivo stability or otherwise enhance its pharmacological properties (e.g., increase the polypeptide's half-life, reduce its toxicity, and / or enhance solubility or uptake). Examples of such modifying or complexing agents include sulfate, gluconate, citrate, and phosphate. The nucleic acid or polypeptide of the composition can also be complexed with molecules that enhance its in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0104] X. Illustrative Embodiments The following aspects are contemplated: All combinations of features and aspects are contemplated. Embodiment 1: A retroviral vector system comprising an envelope plasmid encoding a viral membrane fusion protein of a retrovirus; a packaging plasmid encoding Gag-Pol proteins of said retrovirus; and a transfer plasmid comprising one or more genes of interest for transfer from said retrovirus to a target cell, wherein one or both of said envelope plasmid and said packaging plasmid further encode a binding moiety that binds directly or indirectly to a surface feature of said target cell. Embodiment 2: The embodiment of embodiment 1, wherein said viral membrane fusion protein is an engineered variant of a wild-type viral membrane fusion protein. Embodiment 3: The embodiment of embodiment 2, wherein said engineered variant does not bind to the cognate binding partner of said wild-type viral membrane fusion protein. Embodiment 4: The embodiment of embodiment 2 or 3, wherein said engineered variant is a truncation mutant of said wild-type viral membrane fusion protein. Embodiment 5: The embodiment of any one of embodiments 1 to 4, wherein said binding moiety comprises an extramembrane domain and a transmembrane domain. Embodiment 6: The embodiment of any one of embodiments 1 to 5, wherein the envelope plasmid encodes the binding moiety. Embodiment 7: The embodiment of any one of embodiments 1 to 5, wherein the packaging plasmid encodes the binding moiety. Embodiment 8: The embodiment of any one of embodiments 1 to 7, wherein the binding moiety binds to a ligand, wherein the ligand comprises an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof, and wherein the ligand binds to a surface feature of the target cell. Embodiment 9: The embodiment of embodiment 8, wherein said binding moiety comprises a fluorescein isothiocyanate (FITC) binding domain and said ligand is conjugated to FITC. Embodiment 10: The embodiment of embodiment 9, wherein said binding moiety comprises an anti-fluorescein scFv or a fluorescein-conjugated anticalin. Embodiment 11: The embodiment of embodiment 9 or 10, wherein said ligand is a FITC-conjugated antibody. Embodiment 12: The embodiment of embodiment 8, wherein said binding moiety comprises a biotin-binding domain and said ligand is biotinylated. Embodiment 13: The embodiment of embodiment 12, wherein said binding moiety comprises an anti-biotin scFv, a polyclonal anti-biotin antibody, a monoclonal anti-biotin antibody, a biotin-binding anticalin, or an avidin family protein. Embodiment 14: The embodiment of embodiment 12 or 13, wherein said ligand is a biotinylated antibody. Embodiment 15: The embodiment of any one of embodiments 8 to 14, wherein the target cell surface feature comprises CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4. Embodiment 16: The embodiment of any one of embodiments 1 to 7, wherein the binding moiety directly binds to a surface feature of the target cell. Embodiment 17: The embodiment of embodiment 16, wherein said binding moiety comprises an scFv or a nanobody. Embodiment 18: The embodiment of embodiment 16 or 17, wherein said target cell surface feature comprises CD19 or CD7. Embodiment 19: The embodiment of any one of embodiments 1 to 18, wherein said target cell is a T cell, a B cell, a natural killer (NK) cell, an astrocyte, a dendritic cell (DC), or a monocyte. Embodiment 20: The embodiment of any one of embodiments 1 to 19, wherein said retrovirus is an alpharetrovirus, a gammaretrovirus, or a lentivirus. Embodiment 21: The embodiment of any one of embodiments 1 to 20, wherein the viral membrane fusion protein is a vesicular stomatitis virus G (VSV-G) protein, or is derived from feline endogenous virus RD114, or from baboon endogenous virus BaEV. Embodiment 22: The embodiment of any one of embodiments 1 to 21, wherein the retrovirus is an integrase-deficient retrovirus. Embodiment 23: The embodiment of any one of embodiments 1 to 22, wherein said one or more genes of interest encode a chimeric antigen receptor (CAR), a switch receptor, or a derivative or fragment thereof. Embodiment 24: The embodiment of any one of embodiments 1 to 23, wherein said one or more genes of interest encode a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a recombinase, a flippase, a base editor, a prime editor, a nuclease-impaired Cas, a nuclease-dead Cas, an epigenome editor, a transcription modifier (CRISPRa / i), a transposase, an Argonaute (Ago) protein, an adenosine deaminase acting on RNA (ADAR), a Pumilio RNA-binding family (PUF) protein, a homing endonuclease, or a derivative or fragment thereof. Embodiment 25: The embodiment of any one of embodiments 1 to 24, wherein said one or more genes of interest encode a fluorescent protein, an antibiotic resistance gene, or a derivative or fragment thereof. Embodiment 26: A retroviral packaging cell comprising the retroviral vector system of any one of embodiments 1 to 25. Embodiment 27: The retrovirus, comprising a viral membrane fusion protein; a viral genome comprising one or more genes of interest for transfer from the retrovirus to a target cell; and a binding moiety that binds to a ligand, wherein the ligand comprises an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or derivatives or fragments thereof, and wherein the ligand binds to a surface feature of the target cell. Embodiment 28: The embodiment of embodiment 27, wherein said viral membrane fusion protein is an engineered variant of a wild-type viral membrane fusion protein. Embodiment 29: The embodiment of embodiment 28, wherein said engineered variant does not bind to a cognate binding partner of said wild-type viral membrane fusion protein. Embodiment 30: The embodiment of embodiment 28 or 29, wherein said engineered variant is a truncation mutant of said wild-type viral membrane fusion protein. Embodiment 31: The embodiment of any one of embodiments 27 to 30, wherein said binding moiety comprises an extramembrane domain and a transmembrane domain. Embodiment 32: The embodiment of embodiment 29 or 30, wherein said engineered variant of a wild-type viral membrane fusion protein comprises said binding moiety. Embodiment 33 The embodiment of any one of embodiments 27 to 32, wherein the binding moiety comprises a fluorescein isothiocyanate (FITC) binding domain and the ligand is conjugated to FITC. Embodiment 34: The embodiment of embodiment 33, wherein said binding moiety comprises an anti-fluorescein scFv or a fluorescein-conjugated anticalin. Embodiment 35: The embodiment of embodiment 33 or 34, wherein said ligand is a FITC-conjugated antibody. Embodiment 36: The embodiment of any one of embodiments 27 to 32, wherein the binding moiety comprises a biotin-binding domain and the ligand is biotinylated. Embodiment 37: The embodiment of embodiment 36, wherein said binding moiety comprises an anti-biotin scFv, a biotin-binding anticalin, or an avidin family protein. Embodiment 38: The embodiment of embodiment 36 or 37, wherein said ligand is a biotinylated antibody. Embodiment 39: The embodiment of any one of embodiments 27 to 38, wherein the target cell surface feature comprises CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4. Embodiment 40: The embodiment of any one of embodiments 27 to 39, wherein said retrovirus is an alpharetrovirus, a gammaretrovirus, or a lentivirus. Embodiment 41: The embodiment of any one of embodiments 27 to 40, wherein the viral membrane fusion protein is a vesicular stomatitis virus G (VSV-G) protein, or is derived from feline endogenous virus RD114, or is derived from baboon endogenous virus BaEV. Embodiment 42: The embodiment of any one of embodiments 27 to 41, wherein the retrovirus is an integrase-deficient retrovirus. Embodiment 43: The embodiment of any one of embodiments 27 to 42, wherein said one or more genes of interest encode a chimeric antigen receptor (CAR), a switch receptor, or a derivative or fragment thereof. Embodiment 44: The embodiment of any one of embodiments 27 to 43, wherein said one or more genes of interest encode a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a recombinase, a flippase, a base editor, a prime editor, a nuclease-pathic Cas, a nuclease-inactive Cas, an epigenome editor, a transcription modifier (CRISPRa / i), a transposase, an Argonaute (Ago) protein, an adenosine deaminase acting on RNA (ADAR), a Pumilio RNA-binding family (PUF) protein, a homing endonuclease, or a derivative or fragment thereof. Embodiment 45: A virus-like particle comprising a binding moiety that binds to a ligand, wherein the ligand comprises an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof, and wherein the ligand binds to a surface feature of a target cell. Embodiment 46: The embodiment of embodiment 45, wherein said binding moiety comprises a fluorescein isothiocyanate (FITC) binding domain, and said ligand is conjugated to FITC. Embodiment 47: The embodiment of embodiment 46, wherein said binding moiety comprises an anti-fluorescein scFv or a fluorescein-conjugated anticalin. Embodiment 48: The embodiment of embodiment 46 or 47, wherein said ligand is a FITC-conjugated antibody. Embodiment 49: The embodiment of embodiment 45, wherein said binding moiety comprises a biotin-binding domain and said ligand is biotinylated. Embodiment 50 The embodiment of embodiment 49, wherein said binding moiety comprises an anti-biotin scFv, a biotin-binding anticalin, or an avidin family protein. Embodiment 51: The embodiment of embodiment 49 or 50, wherein said ligand is a biotinylated antibody. Embodiment 52: The embodiment of any one of embodiments 45 to 51, wherein the target cell surface feature comprises CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4. Embodiment 53: The embodiment of any one of embodiments 45 to 52, comprising a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a recombinase, a flippase, a base editor, a prime editor, nuclease-pathic Cas, nuclease-inactive Cas, an epigenome editor, a transcription modifier (CRISPRa / i), a transposase, an Argonaute (Ago) protein, an adenosine deaminase acting on RNA (ADAR), a Pumilio RNA-binding family (PUF) protein, a homing endonuclease, or a derivative or fragment thereof. Embodiment 54: A method for producing the retroviral vector system of any one of embodiments 1 to 25, comprising the step of transfecting a host cell with the retroviral vector system of any one of embodiments 1 to 25. Embodiment 55: A method for preventing or treating a disease in a subject, comprising the step of administering to the subject a retroviral vector system of any one of embodiments 1 to 25, a retroviral packaging cell of embodiment 26, a retrovirus of any one of embodiments 27 to 44, or a virus-like particle of any one of embodiments 45 to 53. Embodiment 56: The embodiment of embodiment 55, wherein the disease comprises a genetic disorder. Embodiment 57: The embodiment of embodiment 56, wherein the disease comprises cancer. Embodiment 58: The embodiment of embodiment 57, wherein the cancer comprises a solid tumor. Embodiment 59: The embodiment of embodiment 55, wherein the disease comprises an infectious disease. Embodiment 60: The embodiment of embodiment 59, wherein the infection is associated with a biomedical device implant in the subject. Embodiment 61: A method for repairing or regenerating damaged or aged tissue in a subject, the method comprising the step of administering to said subject a retroviral vector system of any one of embodiments 1 to 25, a retroviral packaging cell of embodiment 26, a retrovirus of any one of embodiments 27 to 44, or a virus-like particle of any one of embodiments 45 to 53, wherein said target cells comprise induced pluripotent stem cells, embryonic stem cells, adult stem cells, mesenchymal stem cells, or progenitor cells. Embodiment 62: The embodiment of embodiment 61, wherein said damaged or aged tissue comprises a muscle cell, a nerve cell, or a pancreatic cell. [Example]

[0105] The present disclosure will be better understood in consideration of the following non-limiting examples, which are intended for illustrative purposes only and do not limit the scope of the present invention in any way.

[0106] Example 1. Transduction of lymphocytes with retroviruses containing cell-binding domains Retroviruses according to provided embodiments were generated that contain a CD7-targeting nanobody as the cell-binding domain. Wild-type Jurkat cells, which do not naturally express GFP (Figure 3, top panel), were transduced with a traditional lentivirus containing the VSV-G envelope protein and encoding an expression cassette for GFP. Cells transduced with the traditional lentivirus exhibited GFP activity (Figure 3, second panel). The VSV-G of the lentivirus was then mutated to remove its natural binding ability. The mutated lentivirus failed to effectively transduce Jurkat cells (Figure 3, third panel). A pseudotyped retrovirus of a feline endogenous virus (RD114) also failed to transduce the cells (Figure 3, fourth panel). However, the provided retrovirus containing the CD7 cell-binding domain produced transduction results similar to those seen with traditional lentivirus (Figure 3, bottom panel). These results demonstrate effective transduction using the retroviral materials and methods disclosed herein.

[0107] Example 2. Transduction of NK cells with retroviruses carrying cell-binding domains Natural killer (NK) cells from two donors were engineered ex vivo with a chimeric receptor protein based on Toll-like receptor 5 using two different retroviruses. The first retrovirus, a traditional lentivirus bearing a VSV-G envelope protein, demonstrated transduction efficiencies of less than 10% for each of the NK cell populations from the two donors (Figure 4). The second retrovirus, a retrovirus bearing a CD7-binding cell-binding domain according to provided embodiments, demonstrated transduction efficiencies that were 2- to 8-fold higher than those seen with traditional lentiviruses (Figure 4). These results demonstrate the ability of the retroviral materials and methods disclosed herein to effectively transduce therapeutically important cell types that have not been readily transduced with previous retroviral approaches.

[0108] Example 3. Transduction with retroviruses carrying antibody binding domains Universal pseudotyped retroviruses according to provided embodiments were generated by exchanging the binding moiety for a monomeric streptavidin (mSA) biotin-binding domain or an FITC-binding antibody domain (αFITC). Each retrovirus carried an mCherry expression cassette. Jurkat cells were then exposed to various concentrations of retrovirus, where the retrovirus was conjugated to various concentrations of CD71 antibody (αCD71). Results demonstrated that both FITC-conjugated retrovirus (FIG. 6) and biotin-conjugated retrovirus (FIG. 7) transduced Jurkat cells when bound to the antibody. Furthermore, both provided retroviruses were capable of transducing Jurkat cells multiple times, a characteristic indicative of lentiviruses. Additional experiments demonstrated that optimizing both the amount of virus and the concentration of antibody could increase expression of the delivered gene payload, likely through multiple integration events (FIGS. 8 and 9).

[0109] Example 4. Targeting various cellular markers and epitopes with universal pseudotyped retroviruses Universal pseudotyped retroviruses were generated according to the provided embodiments, where the retroviruses contained an FITC-conjugated scFv and an mCherry expression cassette. The retroviruses were used to transduce Jurkat cells engineered with antibodies against lymphocyte markers (CD7) and T cell markers (CD3) (Figure 10), Raji cells engineered with antibodies against B cell markers (CD19 and CD20) (Figure 11), and primary human T cells engineered with antibodies against subtype markers (CD4 and CD8) (Figure 12). Each antibody was tested at three concentrations, and multiple antibody clones were tested for each marker (Figures 10-12). The results demonstrated that universal pseudotyped retroviruses can be effective against multiple targets and multiple epitopes on those targets.

[0110] Example 5. Selective cell targeting with universally pseudotyped retroviruses Universal pseudotyped retroviruses were generated according to the provided embodiments, wherein the retroviruses contained a FITC-conjugated scFv and an mCherry expression cassette. Portions of the retrovirus were conjugated to either CD4 ( FIG. 13 ) or CD8 ( FIG. 14 ) antibodies and delivered to primary human T cells, including both CD4-only and CD8-only T cells. In each of the three donor populations tested, the retrovirus effectively transduced only its intended target, with virtually no off-target cell manipulation observed ( FIGS. 13 and 14 ). These results demonstrate that universal pseudotyped retroviruses as disclosed herein can selectively manipulate specific cell populations in a mixture.

[0111] Example 6. Protein delivery demonstrating in vivo activity in a mouse model The target pseudotyped lentivirus according to the provided embodiment is produced using standard methods. Briefly, a transfer plasmid containing the gene payload of interest, a packaging plasmid, and a modified envelope plasmid, as described in Section III, are transfected into HEK293T cells. The modified envelope plasmid is mutated to eliminate the binding properties of VSV-G and engineered to encode a binding moiety. The binding moiety can be configured or selected to bind directly to a cell marker, or can be configured or selected to bind to a moiety contained in a secondary binding substance, such as a biotin-conjugated antibody or an FITC-conjugated antibody. The virus is isolated and purified. When a secondary binding substance, i.e., a ligand, is used, the purified virus and the secondary binding substance are incubated together for a suitable period of time before administration.

[0112] In a specific example, the payload of interest is GFP, and the binding moiety is either CD7 nanobody (nbCD7) or monomeric streptavidin (mSA).In the case of mSA, virus is incubated with biotin-conjugated CD7 antibody for 30 minutes.In both cases, virus is intravenously injected into mice.After about one week, mouse spleens and peripheral blood are collected and processed to isolate immune cells (spleen cells or peripheral blood mononuclear cells, respectively).Cells are stained for CD7, and the degree of GFP in CD7+ fraction is quantified to confirm on-target efficiency, and simultaneously the GFP in CD7- fraction is quantified to show off-target effect.

[0113] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will recognize that certain changes and modifications can be made within the spirit and scope of the disclosure, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of the various recited embodiments and features can be combined or interchanged in whole or in part. In the foregoing description of various embodiments, those embodiments that refer to separate embodiments may be appropriately combined with other embodiments, as would be recognized by those skilled in the art. Furthermore, those skilled in the art will recognize that the foregoing description is by way of example only and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference were individually incorporated by reference.

Claims

1. an envelope plasmid encoding the viral membrane fusion protein of the retrovirus; a packaging plasmid encoding the Gag-Pol proteins of the retrovirus; and a transfer plasmid containing one or more genes of interest for transfer from the retrovirus to a target cell; A retroviral vector system comprising: The retroviral vector system, wherein one or both of the envelope plasmid and the packaging plasmid further encodes a binding moiety that binds directly or indirectly to a surface feature of the target cell.

2. The retroviral vector system of claim 1, wherein the viral membrane fusion protein is an engineered variant of a wild-type viral membrane fusion protein.

3. The retroviral vector system of claim 2, wherein the engineered variant does not bind to the cognate binding partner of the wild-type viral membrane fusion protein.

4. 4. The retroviral vector system of claim 2 or 3, wherein the engineered variant is a truncated mutant of the wild-type viral membrane fusion protein.

5. The retroviral vector system of any one of claims 1 to 4, wherein the binding moiety comprises an extramembrane domain and a transmembrane domain.

6. The retroviral vector system of any one of claims 1 to 5, wherein the envelope plasmid encodes the binding moiety.

7. The retroviral vector system of any one of claims 1 to 5, wherein the packaging plasmid encodes the binding moiety.

8. 8. The retroviral vector system of any one of claims 1 to 7, wherein the binding moiety binds to a ligand, the ligand comprising an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof, and the ligand binds to a surface feature of the target cell.

9. 9. The retroviral vector system of claim 8, wherein the binding moiety comprises a fluorescein isothiocyanate (FITC) binding domain and the ligand is conjugated to FITC.

10. 10. The retroviral vector system of claim 9, wherein the binding moiety comprises an anti-fluorescein scFv or a fluorescein-conjugated anticalin.

11. 11. The retroviral vector system of claim 9 or 10, wherein the ligand is an FITC-conjugated antibody.

12. 9. The retroviral vector system of claim 8, wherein the binding moiety comprises a biotin-binding domain and the ligand is biotinylated.

13. 13. The retroviral vector system of claim 12, wherein the binding moiety comprises an anti-biotin scFv, a polyclonal anti-biotin antibody, a monoclonal anti-biotin antibody, a biotin-binding anticalin, or an avidin family protein.

14. The retroviral vector system of claim 12 or 13, wherein the ligand is a biotinylated antibody.

15. The retroviral vector system of any one of claims 8 to 14, wherein the target cell surface features include CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.

16. The retroviral vector system of any one of claims 1 to 7, wherein the binding moiety directly binds to a surface feature of the target cell.

17. 17. The retroviral vector system of claim 16, wherein the binding moiety comprises an scFv or a nanobody.

18. 18. The retroviral vector system of claim 16 or 17, wherein the target cell surface features include CD19 or CD7.

19. The retroviral vector system of any one of claims 1 to 18, wherein the target cell is a T cell, a B cell, a natural killer (NK) cell, an astrocyte, a dendritic cell (DC), or a monocyte.

20. The retroviral vector system of any one of claims 1 to 19, wherein the retrovirus is an alpharetrovirus, a gammaretrovirus, or a lentivirus.

21. 21. The retroviral vector system of any one of claims 1 to 20, wherein the viral membrane fusion protein is a vesicular stomatitis virus G (VSV-G) protein, derived from feline endogenous virus RD114, or derived from baboon endogenous virus BaEV.

22. The retroviral vector system of any one of claims 1 to 21, wherein the retrovirus is an integrase-deficient retrovirus.

23. 23. The retroviral vector system of any one of claims 1 to 22, wherein said one or more genes of interest encode a chimeric antigen receptor (CAR), a switch receptor, or a derivative or fragment thereof.

24. 24. The retroviral vector system of any one of claims 1 to 23, wherein said one or more genes of interest encode a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a recombinase, a flippase, a base editor, a prime editor, a nuclease-impaired Cas, a nuclease-dead Cas, an epigenome editor, a transcription modifier (CRISPRa / i), a transposase, an Argonaute (Ago) protein, an adenosine deaminase acting on RNA (ADAR), a Pumilio RNA-binding family (PUF) protein, a homing endonuclease, or a derivative or fragment thereof.

25. 25. The retroviral vector system of any one of claims 1 to 24, wherein said one or more genes of interest encode a fluorescent protein, an antibiotic resistance gene, or a derivative or fragment thereof.

26. A retroviral packaging cell comprising the retroviral vector system of any one of claims 1 to 25.

27. viral membrane fusion proteins; a viral genome containing one or more genes of interest for transfer from the retrovirus to a target cell; and A binding moiety that binds to a ligand, wherein the ligand comprises an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof, and wherein the ligand binds to a surface feature of the target cell. The retrovirus comprising:

28. 28. The retrovirus of claim 27, wherein the viral membrane fusion protein is an engineered variant of a wild-type viral membrane fusion protein.

29. 29. The retrovirus of claim 28, wherein the engineered variant does not bind to the cognate binding partner of the wild-type viral membrane fusion protein.

30. 30. The retrovirus of claim 28 or 29, wherein the engineered variant is a truncated mutant of the wild-type viral membrane fusion protein.

31. The retrovirus of any one of claims 27 to 30, wherein the binding moiety comprises an extramembrane domain and a transmembrane domain.

32. 31. The retrovirus of any one of claims 29 or 30, wherein an engineered variant of said wild-type viral membrane fusion protein comprises said binding moiety.

33. 33. The retrovirus of any one of claims 27 to 32, wherein the binding moiety comprises a fluorescein isothiocyanate (FITC) binding domain and the ligand is conjugated to FITC.

34. 34. The retrovirus of claim 33, wherein the binding moiety comprises an anti-fluorescein scFv or a fluorescein-conjugated anticalin.

35. 35. The retrovirus of claim 33 or 34, wherein the ligand is a FITC-conjugated antibody.

36. 33. The retrovirus of any one of claims 27 to 32, wherein the binding moiety comprises a biotin-binding domain and the ligand is biotinylated.

37. 37. The retrovirus of claim 36, wherein the binding moiety comprises an anti-biotin scFv, a biotin-binding anticalin, or an avidin family protein.

38. 38. The retrovirus of claim 36 or 37, wherein the ligand is a biotinylated antibody.

39. 39. The retrovirus of any one of claims 27 to 38, wherein the target cell surface features comprise CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.

40. 40. The retrovirus of any one of claims 27 to 39, which is an alpharetrovirus, a gammaretrovirus, or a lentivirus.

41. 41. The retrovirus of any one of claims 27 to 40, wherein the viral membrane fusion protein is a vesicular stomatitis virus G (VSV-G) protein, or is derived from feline endogenous virus RD114, or from baboon endogenous virus BaEV.

42. 42. The retrovirus of any one of claims 27 to 41, which is an integrase-deficient retrovirus.

43. 43. The retrovirus of any one of claims 27-42, wherein said one or more genes of interest encode a chimeric antigen receptor (CAR), a switch receptor, or a derivative or fragment thereof.

44. 44. The retrovirus of any one of claims 27-43, wherein said one or more genes of interest encode a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a recombinase, a flippase, a base editor, a prime editor, a nuclease-pathic Cas, a nuclease-inactive Cas, an epigenome editor, a transcription modifier (CRISPRa / i), a transposase, an Argonaute (Ago) protein, an adenosine deaminase acting on RNA (ADAR), a Pumilio RNA-binding family (PUF) protein, a homing endonuclease, or a derivative or fragment thereof.

45. A virus-like particle comprising a binding moiety that binds to a ligand, wherein the ligand comprises an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof, and wherein the ligand binds to a surface feature of a target cell.

46. 46. ​​The virus-like particle of claim 45, wherein the binding moiety comprises a fluorescein isothiocyanate (FITC) binding domain and the ligand is conjugated to FITC.

47. 47. The virus-like particle of claim 46, wherein the binding moiety comprises an anti-fluorescein scFv or a fluorescein-conjugated anticalin.

48. 48. The virus-like particle of claim 46 or 47, wherein the ligand is a FITC-conjugated antibody.

49. 46. ​​The virus-like particle of claim 45, wherein the binding moiety comprises a biotin-binding domain and the ligand is biotinylated.

50. 50. The virus-like particle of claim 49, wherein the binding moiety comprises an anti-biotin scFv, a biotin-binding anticalin, or an avidin family protein.

51. 51. The virus-like particle of claim 49 or 50, wherein the ligand is a biotinylated antibody.

52. 52. The virus-like particle of any one of claims 45 to 51, wherein the surface features of the target cell include CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.

53. 53. The virus-like particle of any one of claims 45-52, comprising a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a recombinase, a flippase, a base editor, a prime editor, a nuclease-disabled Cas, a nuclease-inactive Cas, an epigenome editor, a transcription modifier (CRISPRa / i), a transposase, an Argonaute (Ago) protein, an adenosine deaminase acting on RNA (ADAR), a Pumilio RNA-binding family (PUF) protein, a homing endonuclease, or a derivative or fragment thereof.

54. 26. A method for producing the retroviral vector system of any one of claims 1 to 25, comprising the step of transfecting a host cell with the retroviral vector system of any one of claims 1 to 25.

55. 52. A method for preventing or treating a disease in a subject, comprising administering to the subject a retroviral vector system of any one of claims 1 to 25, a retroviral packaging cell of claim 26, a retrovirus of any one of claims 27 to 44, or a virus-like particle of any one of claims 45 to 53.

56. 56. The method of claim 55, wherein the disease comprises a genetic disorder.

57. 56. The method of claim 55, wherein the disease comprises cancer.

58. 58. The method of claim 57, wherein the cancer comprises a solid tumor.

59. 56. The method of claim 55, wherein the disease comprises an infectious disease.

60. 60. The method of claim 59, wherein the infection is associated with a biomedical device implant in the subject.

61. 1. A method for repairing or regenerating damaged or aged tissue in a subject, comprising: administering to said subject a retroviral vector system of any one of claims 1 to 25, a retroviral packaging cell of claim 26, a retrovirus of any one of claims 27 to 44, or a virus-like particle of any one of claims 45 to 53; The target cells include induced pluripotent stem cells, embryonic stem cells, adult stem cells, mesenchymal stem cells, or progenitor cells; The method.

62. 62. The method of claim 61, wherein the damaged or aged tissue comprises a muscle cell, a nerve cell, or a pancreatic cell.

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